Downregulation Of PTEN Research Articles

Biodegradable FePS3 nanoplatform for efficient treatment of osteosarcoma by combination of gene and NIR-II photothermal therapy

As a common tumor with high incidence, osteosarcoma possesses extremely poor prognosis and high mortality. Improving the survival of osteosarcoma patients is still a great challenge due to the precipice of advancement in treatment. In this study, a combination strategy of gene therapy and photothermal therapy (PTT) is developed for efficient treatment of osteosarcoma. Two-dimensional (2D) FePS3 nanosheets are synthesized and functionalized by poly-L-lysine-PEG-folic acid (PPF) to fabricate a multifunctional nanoplatform (FePS@PPF) for further loading microRNAs inhibitor, miR-19a inhibitor (anti-miR-19a). The photothermal conversion efficiency of FePS@PPF is up to 47.1% under irradiation by 1064 nm laser. In vitro study shows that anti-miR-19a can be efficiently internalized into osteosarcoma cells through the protection and delivery of FePS@PPF nanaocarrier, which induces up-regulation of PTEN protein and down-regulation p-AKT protein. After intravenous injection, the FePS@PPF nanoplatform specifically accumulates to tumor site of osteosarcoma-bearing mice. The in vitro and in vivo investigations reveal that the combined PTT-gene therapy displays most significant tumor ablation compared with monotherapy. More importantly, the good biodegradability promotes FePS@PPF to be cleared from body avoiding potential toxicity of long-term retention. Our work not only develops a combined strategy of NIR-II PTT and gene therapy mediated by anti-miR-19a/FePS@PPF but also provides insights into the design and applications of other nanotherapeutic platforms.

PTEN deficiency facilitates gemcitabine efficacy in cancer by modulating the phosphorylation of PP2Ac and DCK

Gemcitabine is a nucleoside analog that has been successfully used in the treatment of multiple cancers. However, intrinsic or acquired resistance reduces the chemotherapeutic potential of gemcitabine. Here, we revealed a previously unappreciated mechanism by which phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, dominates the decision-making process that is central to the regulation of gemcitabine efficacy in cholangiocarcinoma (CCA). By investigating a gemcitabine-treated CCA cohort, we found that PTEN deficiency was correlated with the improved efficacy of gemcitabine-based chemotherapy. Using cell-based drug sensitivity assays, cell line–derived xenograft, and patient-derived xenograft models, we further confirmed that PTEN deficiency or genetic-engineering down-regulation of PTEN facilitated gemcitabine efficacy both in vitro and in vivo. Mechanistically, PTEN directly binds to and dephosphorylates the C terminus of the catalytic subunit of protein phosphatase 2A (PP2Ac) to increase its enzymatic activity, which further dephosphorylates deoxycytidine kinase (DCK) at Ser 74 to diminish gemcitabine efficacy. Therefore, PTEN deficiency and high phosphorylation of DCK predict a better response to gemcitabine-based chemotherapy in CCA. We speculate that the combination of PP2A inhibitor and gemcitabine in PTEN-positive tumors could avoid the resistance of gemcitabine, which would benefit a large population of patients with cancer receiving gemcitabine or other nucleoside analogs.

Abstract 21: A novel bladder cancer syngeneic mouse model using gene-edited organoids derived from bladder urothelium

Abstract In recent years, immuno-checkpoint inhibitors have expanded the range of treatment options in advanced bladder cancer, but there are still many issues to be overcome, such as drug resistance, effect of combination therapy and prediction of adverse events. To address these issues, there is an urgent need for useful animal models with normal immune system. About half of the human muscle invasive bladder cancers (MIBCs) show p53 mutations in which over 60% are missense mutations. Additionally, the downregulation of tumor suppressor gene PTEN is known in human MIBC. We also investigated cell of origin of chemical-induced mouse bladder cancer using lineage tracing method and found that Krt5 expressing cells with Trp53 alteration efficiently give a rise of high-grade MIBC with squamous differentiation. Based on these results, we have been working on establishing MIBC mouse model using genetically engineered mouse (GEM) in which Cas9 and target gene mutation express specifically in Krt5 expressing cells by Cre-LoxP system. We have also used CRISPR/Cas9 gene editing technique and 3D organoid culture system. We generated organoids from the GEM bladder urothelium with Krt5CreERT2/+; LSL-Cas9; LSL-Trp53R172H/+. For further gene editing, we infected adeno-associated virus with sgRNA to the organoids. The organoid with Trp53R172H/+ and Pten knock-out showed tumorigenicity in immunodeficient mice. The organoid-derived tumors are histologically similar with the basal-squamous subtype of human bladder cancer. Moreover, these tumors exhibited wild type (WT) Trp53 loss of heterozygosity in genomic level. Similarly, we confirmed tumorigenicity in immunodeficient mice with organoids from Trp53flox/flox; Ptenflox/flox and Trp53R172H/flox; Ptenflox/flox mice, but not from Trp53R172H/flox;Pten+/+ mice or Trp53R172H/+; Ptenflox/flox mice, suggesting that both loss of WT Trp53 and Pten loss are required for tumor formation in this setting. RNA sequencing showed Pten loss is associated with up-regulation of metabolism related gene sets and loss of WT Trp53 is associated with up-regulation of proliferation related gene sets. Furthermore, Trp53R172H/flox; Ptenflox/flox organoids showed higher engraftment rate in immunocompetent mice with less infiltration of CD8+ cells compared with Trp53flox/flox; Ptenflox/flox organoids, suggesting that gain of function of mutant Trp53 may affect immune microenvironment. Our data illustrates the usefulness of clinically relevant syngeneic bladder cancer mouse model from GEM-derived organoids for the research of tumor immunity. Citation Format: Akihiro Hamada, Yuki Kita, Takeshi Sano, Ryosuke Ikeuchi, Hideaki Takada, Kenji Nakamura, Toru Sakatani, Takayuki Goto, Atsuro Sawada, Shusuke Akamatsu, Takashi Kobayashi. A novel bladder cancer syngeneic mouse model using gene-edited organoids derived from bladder urothelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 21.

MiR-181b promotes angiogenesis and neurological function recovery after ischemic stroke.

Promotion of new blood vessel formation is a new strategy for treating ischemic stroke. Non-coding miRNAs have been recently considered potential therapeutic targets for ischemic stroke. miR-181b has been shown to promote angiogenesis in hypoxia and traumatic brain injury model, while its effect on ischemic stroke remains elusive. In this study, we found that overexpression of miR-181b in brain microvascular endothelial cells subjected to oxygen-glucose deprivation in vitro restored cell proliferation and enhanced angiogenesis. In rat models of focal cerebral ischemia, overexpression of miR-181b reduced infarction volume, promoted angiogenesis in ischemic penumbra, and improved neurological function. We further investigated the molecular mechanism by which miR-181b participates in angiogenesis after ischemic stroke and found that miR-181b directly bound to the 3'-UTR of phosphatase and tensin homolog (PTEN) mRNA to induce PTEN downregulation, leading to activation of the protein kinase B (Akt) pathway, upregulated expression of vascular endothelial growth factors, down-regulated expression of endostatin, and promoted angiogenesis. Taken together, these results indicate that exogenous miR-181b exhibits neuroprotective effects on ischemic stroke through activating the PTEN/Akt signal pathway and promoting angiogenesis.

Alkannin exerts antitumor properties in cutaneous squamous cell carcinoma by inducing apoptosis and shifting the M1/M2 polarization of tumor-associated macrophages by upregulating PTEN.

Cutaneous squamous cell carcinoma (CSCC) is a common cancer in humans and is the second major type of skin cancer that causes death in humans. In this article, we investigated the effects of alkannin on CSCC progression. We revealed that alkannin curbed CSCC cell viability in a dose-dependent manner and accelerated CSCC cell apoptosis. In addition, alkannin expedited macrophage M1 polarization while curbing M2 polarization. Moreover, alkannin elevated phosphatase and tensin homolog (PTEN) abundance in CSCC cells. The results of bioinformatics analysis revealed that alkannin might modulate CSCC via PTEN. Downregulation of PTEN reversed the effects of alkannin on apoptosis of CSCC cells and M1/M2 polarization of macrophages. Alkannin reduced CSCC tumor growth in a mouse xenograft model. In conclusion, alkannin curbed the advancement of CSCC by expediting apoptosis and facilitating M1 polarization of macrophages by upregulating PTEN. These data may offer a therapeutic approach against CSCC.

Retraction Notice to: miR-221&222 Regulate TRAIL Resistance and Enhance Tumorigenicity through PTEN and TIMP3 Downregulation.

Retraction Notice to: miR-221&222 Regulate TRAIL Resistance and Enhance Tumorigenicity through PTEN and TIMP3 Downregulation.

Transcranial Direct-Current Stimulation Regulates MCT1-PPA-PTEN-LONP1 Signaling to Confer Neuroprotection After Rat Cerebral Ischemia–Reperfusion Injury

Propionic acid (PPA) is a critical metabolite involved in microbial fermentation, which functions to reduce fat production, inhibit inflammation, and reduce serum cholesterol levels. The role of PPA in the context of cerebral ischemia–reperfusion (I/R) injury has yet to be clarified. Increasing evidence indicate that transcranial direct-current stimulation (tDCS) is a safe approach that confers neuroprotection in cerebral ischemia injury. Here, we show that the levels of PPA were reduced in the ischemic brain following a rat cerebral I/R injury and in the cultured rat cortical neurons after oxygen–glucose deprivation (OGD), an in vitro model of ischemic injury. We found that the decreased levels of transporter protein monocarboxylate transporter-1 (MCT1) were responsible for the OGD-induced reduction of PPA. Supplementing PPA reduced ischemia-induced neuronal death after I/R. Moreover, our results revealed that the neuroprotective effect of PPA is mediated through downregulation of phosphatase PTEN and subsequent upregulation of Lon protease 1 (LONP1). We demonstrated that direct-current stimulation (DCS) increased MCT1 expression and PPA level in OGD-insulted neurons, while tDCS decreased the brain infarct volume in the MCAO rats via increasing the levels of MCT1 expression and PPA. This study supports a potential application of tDCS in ischemic stroke.

14P Up-regulated PI3K/mTOR/AKT pathway behind the downregulation of PTEN, FBXW7, genes and miRNA 140-145, ALK mediated chemotherapy resistance in non-small cell lung carcinoma (NSCLC)

Tumor cells have a tendency to proliferate rapidly and can metastasize to other organs of the body. Respiratory carcinomas are among 3rd predominant causes of cancer-related deaths in both genders around the globe. Type of epithelial lung cancer non-small cell lung carcinoma (NSCLC) constitutes 80% of all lung cancer types. The 5-year survival rate of NSCLC is about 16%. This Study was conducted to understand the chemoresistance through expression of proto-onco genes (AKT1, ALK), onco-suppressive (FBXW7, PTEN) genes and their correlation with miRNA 140-145 in NSCLC.

Pten associates with important gene regulatory network to fine-tune Müller glia-mediated zebrafish retina regeneration.

Unlike mammals, zebrafish possess a remarkable ability to regenerate damaged retina after an acute injury. Retina regeneration in zebrafish involves the induction of Müller glia-derived progenitor cells (MGPCs) exhibiting stem cell-like characteristics, which are capable of restoring all retinal cell-types. The induction of MGPC through Müller glia-reprograming involves several cellular, genetic and biochemical events soon after a retinal injury. Despite the knowledge on the importance of Phosphatase and tensin homolog (Pten), which is a dual-specificity phosphatase and tumor suppressor in the maintaining of cellular homeostasis, its importance during retina regeneration remains unknown. Here, we explored the importance of Pten during zebrafish retina regeneration. The Pten gets downregulated upon retinal injury and is absent from the MGPCs, which is essential to trigger Akt-mediated cellular proliferation essential for retina regeneration. We found that the downregulation of Pten in the post-injury retina accelerates MGPCs formation, while its overexpression restricts the regenerative response. We observed that Pten regulates the proliferation of MGPCs not only through Akt pathway but also by Mmp9/Notch signaling. Mmp9-activity is essential to induce the proliferation of MGPCs in the absence of Pten. Lastly, we show that expression of Pten is fine-tuned through Mycb/histone deacetylase1 and Tgf-β signaling. The present study emphasizes on the stringent regulation of Pten and its crucial involvement during the zebrafish retina regeneration.

Exosomes Derived from Adipose Mesenchymal Stem Cells Carrying miRNA-22-3p Promote Schwann Cells Proliferation and Migration through Downregulation of PTEN.

Peripheral nerve injury (PNI) is often resulting from trauma, which leads to severe and permanently disability. Schwann cells are critical for facilitating the regeneration process after PNI. Adipose-derived mesenchymal stem cells (ADSCs) exosomes have been used as a novel treatment for peripheral nerve injury. However, the underlying mechanism remains unclear. In this study, we isolated ADSCs and extracted exosomes, which were verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot (WB). Cocultured with Dorsal Root Ganglion (DRG) and Schwann cells (SCs) to evaluate the effect of exosomes on the growth of DRG axons by immunofluorescence, and the proliferation and migration of SCs by CCK8 and Transwell assays, respectively. Through exosomal miRNA sequencing and bioinformatic analysis, the related miRNAs and target gene were predicted and identified by dual luciferase assay. Related miRNAs were overexpressed and inhibited, respectively, to clarify their effects; the downstream pathway through the target gene was determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and WB. Results found that ADSC-exosomes could promote the proliferation and migration of SCs and the growth of DRG axons, respectively. Exosomal miRNA-22-3p from ADSCs directly inhibited the expression of Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), activated phosphorylation of the AKT/mTOR axis, and enhanced SCs proliferation and migration. In conclusion, our findings suggest that ADSC-exosomes could promote SCs function through exosomal miRNA-22-3p, which could be used as a therapeutic target for peripheral nerve injury.

MicroRNA-382-5p Promotes Oral Squamous Cell Carcinoma Development and Progression by Negatively Regulating PTEN Expression

Oral squamous cell carcinoma (OSCC) local recurrence and distant metastasis remain a poorly understood clinical challenge. The objective of this study was to investigate how dysregulation of miR-382-5p impacts invasion and dissemination of OSCC. Tissue samples were collected from 20 subjects with OSCC. Expression levels of miR-382-5p were determined by quantitative real-time polymerase chain reaction (qRT-PCR), and correlations with clinical characteristics were investigated. qRT-PCR was used to determine the miR-382-5p and peptidyl-prolyl cis/trans isomerase (PTEN) expression in tumor tissues, adjacent normal tissues, normal human oral keratinocyte line, and OSCC line (SCC-9). Cell proliferation, invasion, and migration of knock-in and knock-down miR-382-5p transfectants were assessed using cell counting kit-8 and Transwell assays. PTEN was confirmed to be a downstream target using a TargetScan prediction, dual-luciferase reporter assays, and western blot analysis. Statistical analysis of experimental data was performed with SPSS 22.0 software. We found high expression of miR-382-5p and significant downregulation of PTEN in tumor tissues and SCC-9cells from OSCC patients (P<.05). miR-382-5p expression was lower in early stage (I+II) than in late stage (III+IV), while PTEN exhibited higher expression in early stage (I+II) instead of in late stage (III+IV) (P<.05). In addition, overexpression of miR-382-5p promoted the proliferation, invasion, and migration of OSCC cells. However, the proliferation, invasion, and migration of OSCC cells were inhibited after suppression of miR-382-5p. Finally, PTEN is downregulated by miR-382-5p. MiR-382-5p supports proliferation, invasion, and migration of OSCC cells through the PTEN pathway. Further investigation may improve our understanding of OSCC local recurrence and distant metastasis.

Sirolimus increases the anti-cancer effect of Huai Er by regulating hypoxia inducible factor-1α-mediated glycolysis in hepatocellular carcinoma.

BACKGROUNDGlycolysis caused by hypoxia-induced abnormal activation of hypoxia inducible factor-1α (HIF-1α) in the immune microenvironment promotes the progression of hepatocellular carcinoma (HCC), leading to enhanced drug resistance in cancer cells. Therefore, altering the immunosuppressive microenvironment by imp-roving the hypoxic state is a new goal in improving cancer treatment.AIMTo analyse the role of HIF-1α, which is closely related to tumour proliferation, invasion, metastasis, and angiogenesis, in the proliferation and invasion of liver cancer, and to explore the HIF-1α pathway-mediated anti-cancer mechanism of sirolimus (SRL) combined with Huai Er.METHODSPrevious studies on HCC tissues identified the importance of HIF-1α, glucose transporter 1 (GLUT1), and lactate dehydrogenase A (LDHA) expression. In this study, HepG2 and Huh7 cell lines were treated, under hypoxic and normoxic conditions, with a combination of SRL and Huai Er. The effects on proliferation, invasion, cell cycle, and apoptosis were analysed. Proteomics and genomics techniques were used to analyze the HIF-1α-related signalling pathway during SRL combined with Huai Er treatment and its inhibition of the proliferation of HCC cells.RESULTSHigh levels of HIF-1α, LDHA, and GLUT-1 were found in poorly differentiated HCC, with lower patient survival rates. Hypoxia promoted the proliferation of HepG2 and Huh7 cells and weakened the apoptosis and cell cycle blocking effects of the SRL/Huai Er treatment. This was achieved by activation of HIF-1α and glycolysis in HCC, leading to the upregulation of LDHA, GLUT-1, Akt/mammalian target of rapamycin (mTOR), vascular endothelial growth factor (VEGF), and Forkhead box P3 and downregulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and p27. The hypoxia-induced activation of HIF-1α showed the greatest attenuation in the SRL/Huai Er (S50 + H8) group compared to the drug treatments alone (P < 0.001). The S50 + H8 treatment significantly downregulated the expression of mTOR and HIF-1α, and significantly reduced the expression of VEGF mRNA. Meanwhile, the combined blocking of mTOR and HIF-1α enhanced the downregulation of Akt/mTOR, HIF-1α, LDHA, and GLUT-1 mRNA and resulted in the downregulation of PTEN, p27, and VEGF mRNA (P < 0.001).CONCLUSIONSRL increases the anti-cancer effect of Huai Er, which reduces the promotion of hypoxia-induced HIF-1α on the Warburg effect by inhibition of the PI3K/Akt/mTOR-HIF-1α and HIF-1α-PTEN signalling pathways in HCC.

High-glucose induced toxicity in HK-2 cells can be alleviated by inhibition of miRNA-320c

Diabetic nephropathy (DN) is a major healthcare challenge worldwide. MiRNAs exert a regulatory effect on the progress of DN. Our study proposed to investigate the miR-320c expression and its function on the pathogenesis of DN in vitro. The level of miR-320c in HK-2 cells was quantified by RT-qPCR. Cell morphology, invasion, and migration were observed by optical microscope, Transwell invasion assay, and scratch wound assay. Then, the levels of PTEN, α-SMA, vimentin, E-cadherin, p-PI3K, PI3K, AKT, and p-AKT were analyzed through western blotting. A Dual-luciferase reporter assay was conducted to explore the target relationship between miR-320c and PTEN. It was discovered that miR-320c was over-expressed in high glucose (HG)-treated HK-2 cells. Furthermore, inhibition of miR-320c could alleviate the epithelial-mesenchymal transition (EMT) of HG-induced HK-2 cells and retain the normal morphology of HK-2 cells. Additionally, the miR-320c inhibitor decreased the invasiveness and migration of HG-treated HK-2 cells. Next, the target gene of miR-320c, PTEN, was identified, and the function of miR-320c was reversed by down-regulation of PTEN. Finally, we found inhibition of miR-320c restrained the PI3K/AKT pathway. Therefore, inhibition of miR-320c could alleviate toxicity of HK-2 cells induced by HG via targeting PTEN and restraining the PI3K/AKT pathway, illustrating that miR-320c may act as a new biomarker in the diagnosis of DN.

Cancer-associated fibroblast-derived exosomal microRNA-20a suppresses the PTEN/PI3K-AKT pathway to promote the progression and chemoresistance of non-small cell lung cancer.

BackgroundCancer‐associated fibroblasts (CAFs) contributes to overall tumor progression. In the current survey, we explored the ability of microRNA‐20a (miR‐20a) within these CAF‐derived exosomes to influence non‐small‐cell lung cancer (NSCLC) progression.Materials and methodsNormal tissue‐associated fibroblasts (NAFs) and CAFs were collected from samples of NSCLC patient tumors and paracancerous lung tissues. Exosomes derived from these cells were then characterized via Western blotting, nanoparticle tracking analyses, and transmission electron microscopy. The expression of miR‐20a was assessed via qPCR and fluorescence in situ hybridization (FISH). CCK‐8, EdU uptake, and colony formation assessments were used for evaluating tumor proliferation, while Hoechst staining was performed to monitor the in vitro apoptotic death of tumor cells. A model of xenograft tumor established in nude mice was also used to evaluate in vivo tumor responses.ResultsCAF‐derived exosomes exhibited miR‐20a upregulation and promoted NSCLC cell proliferation and resistance to cisplatin (DDP). Mechanistically, CAF‐derived exosomes were discovered to transmit miR‐20a to tumor cells wherein it was able to target PTEN to enhance DDP resistance and proliferation. Associated PTEN downregulation following exosome‐derived miR‐20a treatment enhanced PI3K/AKT pathway activation.ConclusionThe achieved outcomes explain that CAFs can release miR‐20a‐containing exosomes capable of promoting NSCLC progression and chemoresistance, highlighting this pathway as a possible therapeutic target in NSCLC.

Abstract 6032: Establishment of a bladder cancer model using organoids derived from bladder epithelium in genetically engineered mice

Abstract Despite recent advances in molecular profiling of bladder cancer, molecular mechanisms for urothelial carcinogenesis have not fully understood. It is partly because the lack of faithful disease models that accurately recapitulate human bladder cancer. In our previous study, we have analyzed mutation landscape of chemical-induced mouse bladder cancer and human bladder cancer by exome-sequence and found that some components of histone methyltransferase complex like Kmt2c, Kmt2d, Kdm6a were highly prevalent as common mutations and associated with Trp53. Additionally, the downregulation of tumor suppressor gene PTEN is known in human muscle invasive bladder cancer (MIBC). We also investigated cell of origin of chemical-induced mouse bladder cancer using lineage tracing method and found that Krt5 expressing cells with Trp53 alteration efficiently give a rise of high-grade MIBC with squamous differentiation. Based on these results, we have tried to make MIBC mouse model using genetically engineered mouse in which Cas9 and target gene mutation express specifically in Krt5 expressing cells by Cre-LoxP system, combined with CRISPR/Cas9 gene editing technique and 3D organoid culture system. Eight-week-old mice harboring Krt5CreERT2/+; LSL-Cas9; LSL-Trp53+/R172H were used in this study. After introducing gene recombination by administration of tamoxifen, we generated organoids from bladder epithelial cells. For further gene editing, we infected adeno-associated virus with sgPten and sgMll3 to the organoids, which successfully edited the target genes. There seemed to be no significant differences in growth of the organoids in culture between the wild-type and gene-edited organoids. However, gene-edited organoids showed tumorigenesis in vivo when inoculated in subcutaneous space, renal subcapsular space or bladder submucosa of athymic mice, whereas wild-type organoids did not show tumorigenesis in vivo. Histologically, the organoid-derived tumors showed morphological similarity to human urothelial carcinoma with squamous differentiation and intra-tumoral heterogeneity. Furthermore, tumor derived organoid has a potential of tumor development in immune-competent mice. These tumors also maintain squamous phenotype and tumor heterogeneity. These findings suggest that the technique is a promising approach toward the establishment of a useful model of human bladder cancer for the elucidation of bladder carcinogenesis including driver genes. In addition, we think our syngeneic mouse model may be useful tool for further research of tumor immunity and immune checkpoint inhibitor drugs. Citation Format: Akihiro Hamada, Yuki Kita, Hideaki Takada, Kenji Nakamura, Toru Sakatani, Takeshi Sano, Takayuki Goto, Atsuro Sawada, Shusuke Akamatsu, Takashi Kobayashi. Establishment of a bladder cancer model using organoids derived from bladder epithelium in genetically engineered mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6032.

Abstract 6370: Assessing the effect of blocking CCL2-CCR2 axis on breast cancer brain metastasis

Abstract Among the 300,000 newly diagnosed breast cancer patients every year, 5~20% of them will develop symptomatic brain metastasis, whose 1-year survival rate is lower than 20%. Our group previously reported that brain tumor microenvironment (TME)-mediated PTEN downregulation in brain metastatic tumor cells increases the secretion of the chemokine CCL2, which enhances the infiltration of CCR2+ bone marrow derived myeloid cells, eventually promoting brain metastasis outgrowth. However, the mechanism of CCL2-CCR2 axis promoting brain metastasis is not clear, and the efficacy of targeting either CCR2 or CCL2 remained to be assessed. Here we show that Ccr2 antagonism significantly blocked Ccr2high myeloid cells trafficking in the blood and infiltration into the brain TME. However, Ccr2 antagonism did not sufficiently impede breast cancer brain metastasis in multiple preclinical models. Nevertheless, knocking down CCL2 or targeting CCL2 exhibited significant inhibition of brain metastasis growth. Subsequent analysis of the brain TME through mass cytometry demonstrated dramatically decreased infiltration of both Ccr2high and Ccr2low myeloid cell populations in the CCL2 targeted group, implying that the Ccr2low myeloid cells are also involved in promoting brain metastasis growth. Our data highlights the differential responses between targeting CCR2 or CCL2 in multiple preclinical brain metastasis models, which direct us to explore the function of Ccr2low myeloid cells in regulating brain metastasis progression. Our findings also suggest that carefully choosing between CCR2 or CCL2 as a target to control brain metastasis is important in clinical practice. Citation Format: Yimin Duan, Yutao Qi, Lin Zhang, Xiangliang Yuan, Dihua Yu. Assessing the effect of blocking CCL2-CCR2 axis on breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6370.

Synthesis of 3-N-/O-/S-methyl-imidazo[1,2-a] pyridine derivatives for caspase-3 mediated apoptosis induced anticancer activity

A library of 49 analogs of imidazo[1,2-a]pyridine with 2-halo, aryl, styryl and phenylethynyl-substitution at C-2 position and N-/O-/S-methyl linkage at C-3 position, have been synthesized and evaluated for their anti-proliferative activity against breast (MCF-7, MDA-MB-231), pancreatic (MiaPaca-2), lung (A549), prostate (PC-3) and colon (HCT-116) cancer cell lines and normal cells (HEK-293). Among the screened compounds, 5b exhibited best anticancer potential in all tested cancer cells with IC50 ranging from 3.5 to 61.1 µM and no toxicity in normal cells. Further, mechanistic study of 5b revealed concentration dependent increased generation of ROS, reduced mitochondrial membrane potential (MMP), surface and nuclear morphological alterations and inhibition of colony formation in HCT-116 cells. Western blot results had shown that the cell death in HCT-116 colon cancer cells was achieved through the induction of apoptosis via upregulation of the PTEN gene and downregulation of AKT pathway. Similarly, 5b treatment induced caspase-3 cleavage which is a hallmark of apoptosis. Molecular docking and binding energy (ΔG) studies of hit 5b with respect to three important cancer targets (EGFR, mTOR and PI3Kα) revealed strong binding of inhibitor with PI3Kα (docking score −6.932 and ΔG −56.297).

Epigenetic Silencing of PTEN and Epi-Transcriptional Silencing of MDM2 Underlied Progression to Secondary Acute Myeloid Leukemia in Myelodysplastic Syndrome Treated with Hypomethylating Agents.

In myelodysplastic syndrome (MDS), resistance to hypomethylating agents (HMA) portends a poor prognosis, underscoring the importance of understanding the molecular mechanisms leading to HMA-resistance. In this study, P39 and Kasumi-1 cells and their azacitidine-resistant and decitabine-resistant sublines were evaluated comparatively with transcriptomic and methylomic analyses. Expression profiling and genome-wide methylation microarray showed downregulation of PTEN associated with DNA hypermethylation in P39 cell lines resistant to azacitidine and decitabine. This pattern of PTEN dysregulation was also confirmed in a cohort of patients failing treatment with HMA. DNA hypomethylation of MDM2 was detected with downregulation of MDM2 in HMA resistant cell lines. Long-read sequencing revealed significant RNA hypomethylation of MDM2 resulting in alternative splicing and production of a truncated MDM2 transcript in azacitidine-resistant P39 cells. The expression of this MDM2 truncated transcript was also significantly increased in HMA-resistant patients compared with HMA-responsive patients. In conclusion, epigenetic and epi-transcriptomic dysregulation of PTEN and MDM2 were associated with resistance to hypomethylating agents.

DNMT3a negatively regulates PTEN to activate the PI3K/AKT pathway to aggravate renal fibrosis

BackgroundRenal fibrosis has become one of the major diseases threatening global public health and harming human life and health. PTEN methylation plays an important role in fibrotic diseases of many organs. However, the relationship between PTEN methylation and renal fibrosis is still elusive. MethodsIn the present study, we established a unilateral ureteral obstruction (UUO) mouse model in vivo and a transforming growth factor β1 (TGF-β1)-stimulated renal tubular epithelial cell (HK−2) model in vitro. The degree of renal interstitial fibrosis was detected by haematoxylin-eosin (HE) staining and Masson's trichrome staining. Western blot (WB), qRT–PCR, immunohistochemistry (IHC) and methylation-specific PCR (MSP) analyses were used to determine the mechanism by which PTEN methylation regulates renal fibrosis. The α-SMA fibrosis marker was detected by immunofluorescence (IF). Additionally, the relationship of PTEN and DNMT3a in UUO was determined by ChIP-qRT–PCR. ResultsOur results showed that the promoter region of PTEN was methylated in UUO. Compared to the sham group, the expression of PTEN was significantly reduced in the UUO group. However, the demethylation reagent significantly inhibited epithelial-mesenchymal transition (EMT), which showed increased expression of E-cadherin and decreased expression of α-SMA and fibronectin. Moreover, treatment of HK-2 cells with 5-aza-dc reversed the activation of the TGF-β1-induced PI3K/AKT signalling pathway, which inhibited renal fibrosis. WB analysis demonstrated that TGF-β1 inhibited the PTEN protein expression level and DNMT3a knockdown reversed the inhibitory effect of TGF-β1 on PTEN expression. Furthermore, ChIP-qRT–PCR showed that DNMT3a interacted with PTEN. Finally, we found that DNMT3a negatively regulated PTEN to activate the PI3K/AKT signalling pathway and aggravate renal fibrosis in vitro and in vivo. ConclusionIn summary, these results indicated that renal fibrosis is related to the downregulation of PTEN. Additionally, DNMT3a negatively regulates PTEN to activate the PI3K/AKT signalling pathway and induce EMT in renal tubular epithelial cells, thereby aggravating renal fibrosis.

Analysis of the lncRNA-miRNA-mRNA Network Reveals a Potential Regulatory Mechanism of EGFR-TKI Resistance in NSCLC.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are widely used for patients with EGFR-mutated lung cancer. Despite its initial therapeutic efficacy, most patients eventually develop drug resistance, which leads to a poor prognosis in lung cancer patients. Previous investigations have proved that non-coding RNAs including long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs) contribute to drug resistance by various biological functions, whereas how they regulate EGFR-TKI resistance remains unclear. In this study, we examined gene expression using the microarray technology on gefitinib-resistant NSCLC cells to obtain differentially expressed (DE) lncRNAs and mRNAs. A total of 45 DE-lncRNAs associated with overall survival and 1799 target DE-mRNAs were employed to construct a core lncRNA–miRNA–mRNA network to illustrate underlying molecular mechanisms of how EGFR-TKI resistance occurs in NSCLC. We found that target DE-mRNAs were mainly enriched in pathways involved in EGFR-TKI resistance, especially the target DE-mRNAs regulated by LINC01128 were significantly enriched in the PI3K/Akt signaling pathway, where the synergy of these target DE-mRNAs may play a key role in EGFR-TKI resistance. In addition, downregulated LINC01128, acting as a specific miRNA sponge, decreases PTEN via sponging miR-25-3p. Furthermore, signaling reactions caused by the downregulation of PTEN would activate the PI3K/Akt signaling pathway, which may lead to EGFR-TKI resistance. In addition, a survival analysis indicated the low expression of LINC01128, and PTEN is closely related to poor prognosis in lung adenocarcinoma (LUAD). Therefore, the LINC01128/miR-25-3p/PTEN axis may promote EGFR-TKI resistance via the PI3K/Akt signaling pathway, which provides new insights into the underlying molecular mechanisms of drug resistance to EGFR-TKIs in NSCLC. In addition, our study sheds light on developing novel therapeutic approaches to overcome EGFR-TKI resistance in NSCLC.